Tissue container systems

ABSTRACT

The present invention relates generally to tissue container systems that find use in the transport of tissues and methods of using the tissue container systems. In particular the present invention relates to systems that support the transport, thawing and use of cryopreserved human skin equivalents, and methods of their use by a health care provider.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/481,405, filed Jul. 26, 2019, which claims the benefit ofInternational Application Number PCT/US2018/015490, filed Jan. 26, 2018,which claims the benefit of U.S. provisional application number62/451,379, filed Jan. 27, 2017, all of which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to tissue container systems thatfind use in the transport of tissues and methods of using the tissuecontainer systems. In particular the present invention relates tosystems that support the transport, thawing and use of cryopreservedhuman skin equivalents, and methods of their use by a health careprovider.

BACKGROUND OF THE INVENTION

A major impediment to the acceptance of engineered tissues by medicalpractitioners, healthcare providers, and second party payers is the lackof a means to effectively and efficiently preserve and store engineeredtissues. The nature of living cells and tissue products makesdevelopment of long-term storage challenging. Current engineered tissuesmust often be stored and shipped under carefully controlled conditionsto maintain viability and function. Typically, engineered tissueproducts take weeks or months to produce but must be used within hoursor days after manufacture. As a result, tissue engineering companiesmust continually operate with their production facilities at topcapacity and absorb the costs of unsold product which must be discarded.As one specific example, APLIGRAF requires about four weeks tomanufacture, is usable for only 15 days and must be maintained between20 and 23° C. until used. As another example, EPICEL is transported by anurse from Genzyme Biosurgery's production facility in Cambridge, Mass.to the point of use in a portable incubator and is used immediately uponarrival. Such constraints represent significant challenges to developingconvenient and cost-effective products.

Cryopreservation has been explored as a solution to the storage problem,but it is known to induce tissue damage through ice formation, chillinginjury, and osmotic imbalance. Besides APLIGRAF, the only other approvedfull-thickness living skin equivalent, ORCEL, has been evaluated as afrozen product but had the drawback that it must be maintained attemperatures below −100° C. prior to use. This requires specializedproduct delivery and storage conditions, including use of liquidnitrogen for storage, which is expensive and not readily available inrural clinics and field hospitals.

Accordingly, what is needed in the art are improved methods ofcryopreserving viable engineered tissues and cells for storage underconditions that are routinely available at the point of use.

SUMMARY OF THE INVENTION

The present invention relates generally to tissue container systems thatfind use in the transport of tissues and their subsequent use by ahealth care provider, and in particular to systems that support thetransport, thawing and use of cryopreserved human skin equivalents.

Accordingly, in some embodiments, the present invention provides tissuecontainers comprising: a perimeter wall and a substantially planarbottom surface defining a dish, the perimeter wall having a male end anda female end, the male end of the perimeter wall having projectingtherefrom a ridge having a length and width, wherein the female end ofthe perimeter wall defines a space corresponding to the length and widthof the ridge so that when an identical tissue container is placed on topof the tissue container the female end of the tissue containerreleasably receives the ridge extending from the male end of theidentical tissue container, and the bottom surface having a perimeterand comprising a perimeter ledge extending around the perimeter toprovide a reservoir defined by the perimeter ledge and the bottomsurface. In some embodiments, the perimeter wall has a flange extendingtherefrom. In some embodiments, the flange comprises one or more tabsextending from the male end of the perimeter wall. In some embodiments,the flange comprises one or more tabs extending from the female end ofthe perimeter wall. In some embodiments, the ridge has a proximal endand the proximal end of the ridge has one or more indents therein.

In some embodiments, the present invention provides tissue containerassemblies comprising: substantially identical top and bottom tissuecontainers, each of the top and bottom tissue containers comprising aperimeter wall and a substantially planar bottom surface defining adish, the bottom surface having a perimeter and comprising a perimeterledge extending around the perimeter to provide a reservoir defined bythe perimeter ledge and the bottom surface, and the perimeter wallhaving a male end and a female end, the male end of the perimeter wallhaving projecting therefrom a ridge having a length and width, whereinthe female end of the perimeter wall defines a space corresponding tothe length and width of the ridge so that when the top tissue containeris placed on the bottom tissue container the female end of the bottomtissue container releasably receives the ridge extending from the maleend of the top tissue container. In some embodiments, the perimeter wallof the top tissue container has a top flange extending therefrom and theperimeter wall of the bottom tissue container has a bottom flangeextending therefrom so that when the top and bottom tissue containersare assembled the top and bottom flanges contact one another. In someembodiments, the top flange comprises one or more tabs extending fromthe male end of the perimeter wall and one or more tabs extending fromthe female end of the perimeter wall. In some embodiments, the bottomflange comprises one or more tabs extending from the male end of theperimeter wall and one or more tabs extending from the female end of theperimeter wall. In some embodiments, the bottom flange comprises one ormore tabs extending from the male end of the perimeter wall and one ormore tabs extending from the female end of the perimeter wall and thewherein the top flange comprises one or more tabs extending from themale end of the perimeter wall and one or more tabs extending from thefemale end of the perimeter wall so that when the top and bottom tissuecontainers are assembled the tabs are offset.

In some embodiments, the present invention provides tissue containersystems comprising: substantially identical top and bottom tissuecontainers and a tray comprising a porous bottom surface, each of thetop and bottom tissue containers comprising a perimeter wall and asubstantially planar reservoir bottom surface defining a dish, thereservoir bottom surface having a perimeter and comprising a perimeterledge extending around the perimeter to provide a reservoir defined bythe perimeter ledge and the reservoir bottom surface, wherein the trayis sized to be supported by the ledge and above the reservoir bottomsurface when inserted into the tissue container, and the perimeter wallhaving a male end and a female end, the male end of the perimeter wallhaving projecting therefrom a ridge having a length and width, whereinthe female end of the perimeter wall defines a space corresponding tothe length and width of the ridge so that when the top tissue containeris placed on the bottom tissue container the female end of the bottomtissue container releasably receives the ridge extending from the maleend of the top tissue container. In some embodiments, the perimeter wallof the top tissue container has a top flange extending therefrom and theperimeter wall of the bottom tissue container has a bottom flangeextending therefrom so that when the top and bottom tissue containersare assembled the top and bottom flanges contact one another. In someembodiments, the top flange comprises one or more tabs extending fromthe male end of the perimeter wall and one or more tabs extending fromthe female end of the perimeter wall. In some embodiments, the bottomflange comprises one or more tabs extending from the male end of theperimeter wall and one or more tabs extending from the female end of theperimeter wall. In some embodiments, the bottom flange comprises one ormore tabs extending from the male end of the perimeter wall and one ormore tabs extending from the female end of the perimeter wall and thewherein the top flange comprises one or more tabs extending from themale end of the perimeter wall and one or more tabs extending from thefemale end of the perimeter wall so that when the top and bottom tissuecontainers are assembled the tabs are offset. In some embodiments, theporous bottom surface of the tray is a porous membrane. In someembodiments, the ridge has a proximal end and the proximal end of theridge has one or more indents therein and the tray has one or more traytabs so that when the tray is inserted into the bottom tissue containerthe one or more tabs are inserted into the one or more indents. In someembodiments, the systems further comprise a tissue supported on theporous bottom surface of the tray. In some embodiments, the tissue iscryopreserved. In some embodiments, the tissue is an organotypic skinsubstitute. In some embodiments, the systems further comprise a sterilepackage containing the tissue container system. The tissue containersystem can be provided as a kit with one or more absorbent medium and/orone or more liquid media, such as a tissue compatible solution.

In some embodiments, the present invention provides methods of providinga tissue for use by a health care provider comprising packaging a tissuein the tissue container system of the preceding paragraph and providingthe packaged tissue to a health care provider in need thereof. In someembodiments, the present invention provides methods of thawing acryopreserved tissue comprising: providing a cryopreserved tissue in thetissue container system as described above, removing the top tissuecontainer to expose the cryopreserved tissue, optionally transferringthe cryopreserved tissue to a new container system, and filling thereservoir in the bottom tissue container with a liquid medium underconditions that the cryopreserved tissue thaws to provide a thawedtissue. In some embodiments, the cryopreserved tissue is an organotypichuman skin substitute. In some embodiments, the methods further compriseapplying or grafting the organotypic human skin substitute to a burn ora wound on a patient in need thereof.

In some embodiments, the present invention provides a tissue container100 shown in FIG. 1 that comprises a perimeter wall 105 and asubstantially planar bottom surface 110 defining a dish. The perimeterwall 105 has a male end 115 and a female end 120. The male end 115 ofthe perimeter wall 105 has a ridge 125 extending therefrom that has alength and a width. The female end 120 of the perimeter wall 105 definesa space 130 corresponding to the length and width of the ridge 125 sothat when an identical tissue container is placed on top of the tissuecontainer 100 the space 130 provided in said female end 120 of thetissue container can releasably receive the ridge 125 extending from themale end of the identical tissue container as shown in more detailbelow. The bottom surface 110 comprises a perimeter ledge 135 extendingaround the perimeter of the bottom surface 110. The perimeter ledge 135forms a reservoir 140 on the bottom of the container that is preferablyabout 0.50 to 1.5 mm deep, and most preferably about 0.75 mm deep andwhich can be filled with a liquid medium. The perimeter wall 105preferably has a flange 145 extending therefrom. In some embodiments,the tissue container 100 further comprises (a) a flange 145 comprisingone or more tabs 150 extending the male end 115 and female end 120 ofthe perimeter wall, (b) a ridge 125 that has one more indents 155therein that are configured to receive tabs on a tray, (c) a perimeterwall 105 comprising a plurality of grip projections 160, preferablypositioned on the male end 115 of the perimeter wall 105, or (d) anycombination thereof. The present invention also provides a tissuecontainer assembly comprising substantially identical bottom and topcontainers, wherein the bottom and top containers are a tissue containerdescribed in this paragraph. The present invention also provides atissue container system shown in FIG. 4 comprising a tissue containerassembly of this paragraph and a tray 410. The tray is sized so that itrests on top of the perimeter ledge on the bottom surface of the bottomcontainer as described above. The tray 410 comprises sidewalls 415. Tabs420 extend from the sidewalls 415 so that they engage and are insertedinto indents 425 in the ridge 430 on the male end 435 of the bottomcontainer 405. The tray has a porous bottom surface 440, which isoptionally a porous membrane. An identical top container can be placedon the bottom container and closed, without interference from thecontained tray. The tissue container system can be optionally sealed,preferably heat sealed, in a sterile bag to provide a primary package.The primary package can be optionally sealed inside a secondary bag. Thetissue container system or package containing the tissue containersystem can be provided as a kit with one or more absorbent medium and/orone or more liquid media, such as a tissue compatible solution.

In some embodiments, the present invention provides methods of providinga tissue for use by a health care provider comprising packaging a tissuein the tissue container system as described in the preceding paragraphand providing the packaged tissue to a health care provider in needthereof. In some embodiments, the present invention provides methods ofproviding a tissue for use to treat a wound or a burn comprisingpackaging a tissue in the tissue container system as described in thepreceding paragraph and providing the packaged tissue to a health careprovider for use to treat wound or a burn. In some embodiments, thepresent invention provides a method of thawing a cryopreserved skinequivalent prior to application to a subject. The method comprisesproviding a cryopreserved tissue, preferably an organotypically culturedskin equivalent, in a tissue container system as described in thepreceding paragraph, removing the top tissue container to expose thecryopreserved tissue, and filling the reservoir in the bottom tissuecontainer with a liquid medium under conditions that the cryopreservedtissue thaws to provide a thawed tissue, where the cryoprotectantcontained within the tissue is diluted into the liquid medium, leaving atissue that is substantially free of cryoprotectant. In otherembodiments, the method comprises removing a primary or secondarypackage containing a tissue container system comprising a cryopreservedtissue from a freezer or shipping container, removing the tissuecontainer system from the package(s), removing the top tissue containerto expose the cryopreserved tissue, and transferring the tray with thecryopreserved skin equivalent from the first tissue container into asecond tissue container that is sterile and staged in the sterile fieldand contains a liquid medium in the container reservoir, such that thetransferred cryopreserved tissue thaws to provide a thawed tissue andthe cryoprotectant contained within the tissue is diluted into theliquid medium. In some of the above embodiments, the liquid medium is atissue compatible solution, preferably a buffered solution. In stillother embodiments, the tray with the cryopreserved skin equivalent isremoved from the tissue container and placed on an absorbent medium toremove thawed cryoprotectant solution from the skin equivalent. Theabsorbent medium may be in any suitable, preferably sterile, vessel(e.g., a culture vessel or a fresh tissue container assembly). Thepresent invention is not limited to the use of a particular absorbentmedium. The absorbent medium preferably comprises a tissue-compatiblesolution.

Other aspects and iterations of the invention are described morethoroughly below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a perspective view of a tissue container in accordance withone embodiment.

FIG. 2 is disassembled perspective view of a tissue container assemblyaccording to one embodiment.

FIG. 3 is a perspective view of an assembled tissue container assemblyaccordingly to one embodiment.

FIG. 4 is a perspective view of a tissue container with an inserted trayaccording to one embodiment.

FIG. 5 is a graph of tissue viability after 1-day re-culture. Data aremean±stdev of 15 samples per group (5 samples/tissue×3 tissues/conditionin each batch).

FIG. 6 is a graph of post-thaw VEGF secretion during 1-day re-culture.Data are mean±stdev of 3 tissues per condition in each batch.

FIG. 7A and FIG. 7B are graphs of post-thaw tissue barrier functionafter 1-day re-culture with initial DPM (FIG. 7A) and DPM change (FIG.7B). Data are mean±stdev of 12 reads per group (4 samples/tissue×3tissues/condition in each batch).

DETAILED DESCRIPTION

The present invention relates generally to tissue container systems thatfind use in the transport of tissues and their subsequent use by ahealth care provider, and in particular to systems that support thetransport, thawing and use of cryopreserved human skin equivalents.

As used herein, the terms “skin equivalent,” “human skin equivalent,”“human skin substitute,” and “organotypic human skin equivalent” areused interchangeably to refer to an in vitro derived culture ofkeratinocytes that has stratified into squamous epithelia. Typically,the skin equivalents are produced by organotypic culture and include adermal layer in addition to a keratinocyte layer.

As used herein, the term “sterile” refers to a skin equivalent that isessentially or completely free of detectable microbial or fungalcontamination.

As used herein, the term “NIKS cells” refers to cells having thecharacteristics of the cells deposited as cell line ATCC CRL-12191.“NIKS” stands for near-diploid immortalized keratinocytes and is aregistered trademark.

As used herein, the term “viable” when used in reference to a skinequivalent refers to the viability of cells in the skin equivalentfollowing cryopreservation. In preferred embodiments, a “viable” skinhas an A550 of at least 50%, 60%, 70%, 80% or 90% of a controlnon-cryopreserved tissue as measured by an MTT assay or at least 50%,60%, 70%, 80% or 90% of the readout value of a similar viability assay.

As used herein, the term “culture vessel” refers to any vessel of thetype commonly used to culture cells or tissues and includes circular,rectangular, and square dishes formed from a suitable material such astissue culture plastic, polystyrene, polymers, plastics, glass, etc. Theterm “culture vessel” and “growth chamber” are used interchangeably.Tissue containers of the present disclosure are not culture vessels, asused herein, at least because the tissue containers of the presentdisclosure are not of a suitable size for long-term culture.

The tissue containers of the instant invention make efficient use offreezer and surgical suite space as they are approximately 60% smallerthan previously utilized containers. The tissue containers arecompatible with a tray that includes a porous membrane as bottom surfaceupon which a tissue (e.g., an organotypic skin substitute) can besupported. The other surfaces of the tray are preferably clear ortranslucent plastics produced by a thermoforming process from a plasticsheet, injection molding, or other methods known in the art tomanipulate plastics. Suitable plastics include medical grade plastics,for example, polyethylene terephthlate glycol-modified (PETG),polystyrene, etc. In some preferred embodiments, the tray is apreferably a tray as described in paragraph [0030] herein. The tissuecontainers include a reservoir that can be filled with media to thaw thetissue in the container and remove cryoprotectant when the tissue hasbeen frozen. This provides an advantage over previous systems used forthawing tissues where the tissue had to be removed from the container inthe surgical sterile field and then placed on a Telfa® pad. The tissuecontainers of the present invention preferably include a top and bottomwhich are mirror images of one another. The top and bottom pieces of thecontainer assembly are substantially identical and can be snappedtogether to form an enclosed container. The use of a top and bottomwhich are substantially identical means that both the top and bottompiece can be produced from the same molds, which creates efficienciesduring the production of the top and bottom pieces. The top and bottompieces are preferably clear and produced by a thermoforming process froma plastic sheet. Suitable plastics include medical grade thermoformableplastics, for example, polyethylene terephthlate glycol-modified (PETG).Accordingly, the present invention provides improved tissue containersand tissue container systems which will be described in more detailbelow.

FIG. 1 shows a tissue container 100. In some embodiments, the tissuecontainer 100 preferably comprises a perimeter wall 105 and asubstantially planar bottom surface 110 defining a dish. The perimeterwall 105 has a male end 115 and a female end 120. The male end 115 ofthe perimeter wall 105 has a ridge 125 extending therefrom that has alength and a width. The female end 120 of the perimeter wall 105 definesa space 130 corresponding to the length and width of the ridge 125 sothat when an identical tissue container is placed on top of the tissuecontainer 100 the space 130 provided in said female end 120 of thetissue container can releasably receive the ridge 125 extending from themale end of the identical tissue container as shown in more detailbelow. The bottom surface 110 comprises a perimeter ledge 135 extendingaround the perimeter of the bottom surface 110. The perimeter ledge 135forms a reservoir 140 on the bottom of the container that is preferablyabout 0.50 to 1.5 mm deep, and most preferably about 0.75 mm deep andwhich can be filled with a liquid medium. The perimeter wall 105preferably has a flange 145 extending therefrom. In some embodiments,the flange 145 comprises one or more tabs 150 extending the male end 115and female end 120 of the perimeter wall. In some embodiments, the ridge125 has one or more indents 155 therein that are configured to receivetabs on a tray, which is shown in more detail below. In some furtherembodiments the perimeter wall 105 preferably comprises a plurality ofgrip projections 160, preferably positioned on the male end 115 of theperimeter wall 105.

FIG. 2 shows an expanded view of a tissue container assembly 200 of theinstant invention. The tissue container assembly 200 preferablycomprises substantially identical bottom and top containers 205 and 210.Each of the bottom and top containers 205 and 210 comprise a perimeterwall 215 and 220 and have a bottom surface 225 in the case of the bottomcontainer 205 and a top surface 230 in the case of the top container210. The bottom surface 225 comprises a perimeter ledge 235 extendingaround the perimeter of the bottom surface 225. The perimeter ledge 235forms a reservoir 240 on the bottom of the bottom container 205 that ispreferably about 0.50 to 1.5 mm deep, and most preferably about 0.75 mmdeep and which can be filled with a liquid medium. Each of the bottomand top containers 205 and 210 comprise male and female ends 245 and250. The male ends 245 have a ridge 255 extending therefrom that has alength and a width. The female ends 250 define a space 260 correspondingto the length and width of the ridges 255 so that when the top container210 is placed on the bottom container 205 along the alignment shown bydashed lines 265 the space 260 provided in said female ends 250 of thebottom and top tissue containers 205 and 210 can releasably receive theridges 255 so that the bottom and top containers 205 and 210 can bereleasably snapped together. The perimeter walls 215 and 220 preferablyhave flanges 270 and 275 extending therefrom. In some embodiments, theflanges comprise one or more tabs 280 extending the male and female ends245 and 250. In some embodiments, the ridges 255 have one or moreindents 285 therein that are configured to receive tabs on a tray, whichis shown in more detail below. In some further embodiments the perimeterwalls preferably comprises a plurality of grip projections 290,preferably positioned on the male ends 245. FIG. 3 shows a containerassembly 300 of the present invention where the bottom container 305 andtop container 310 are fully engaged to form an enclosed container.

The present invention further provides a tissue container systemcomprising the bottom and top containers described above along with atray. FIG. 4 shows a bottom container of the present invention intowhich a tray 410 has been inserted. The tray 410 is sized so that itrests on top of the perimeter ledge on the bottom surface of the bottomcontainer as described above. The tray 410 comprises sidewalls 415. Tabs420 extend from the sidewalls 415 so that they engage and are insertedinto indents 425 in the ridge 430 on the male end 435 of the bottomcontainer 405. Sidewalls 415 and tabs 420 are preferably clear ortranslucent plastics produced by a thermoforming process from a plasticsheet, injection molding, or other methods known in the art tomanipulate plastics. Preferred plastics are medical grade thermoformableplastics including, but not limited to, polyethylene terephthlateglycol-modified (PETG) and polystyrene. In some preferred embodiments,the plastic used for sidewalls 415 and tabs 420 is polystyrene. The traypreferably has a porous bottom surface 440. In some preferredembodiments, the porous bottom surface is a porous membrane, preferablya semi-permeable polymer film, more preferably a semi-permeabletrack-etched polymer film. The membrane can be tissue culture treated(e.g., plasma treated) to improve cell attachment. In furtherembodiments, the membrane has a nominal thickness of at least 5 microns,in some example, about 5 microns to about 20 microns, preferably about10 microns to about 20 microns, more preferably about 10 microns toabout 15 microns. In other examples, the membrane has a nominalthickness of about 10 microns. Suitable membrane materials are known inthe art and include, but are not limited to, polyethylene terephthalate,polyester, polycarbonate, or any other membrane material used incommercially available, tissue-culture treated inserts (e.g.,Transwell®, Snapwell™, etc.) with a multiplicity of open porestherethrough. Preferably the pores have a nominal pore size of about 0.1micron to about 10 microns, preferably about 0.1 micron to about 0.8micron, more preferably about 0.2 micron to about 0.8 micron, even morepreferably about 0.4 micron, about 0.5 micron, or about 0.6 micron. Themembrane preferably has a nominal pore density between about 1×10⁸ andabout 4×10⁸ pores per square centimeter, though a wider range is alsoacceptable. Most preferably, a membrane is formed from polycarbonatehaving pores with a nominal size of about 0.4 micron and a nominal poredensity about 1×10⁸ pores per square centimeter. The membrane may beattached to sidewalls 415 by any suitable method known in the art, forexample by heat sealing, sonic welding, solvent bonding, adhesivebonding and the like.

The present invention may be used to cryopreserve, store and/ortransport a variety of tissues. The tissues are preferably supported onthe porous bottom surface of the tray and are enclosed with a containerassembly of the present invention comprising bottom and top containers.In some preferred embodiments, the tissues are cryopreserved. In someembodiments, the tissues are skin tissues, for example, cadaver skin ororganotypic skin equivalents. In some exemplary embodiments, the tissuesare organotypic skin equivalents or cryopreserved organotypic skinequivalents.

The present invention is not limited to any particular organotypic skinequivalent. Indeed, the present invention contemplates the use of avariety of cell lines and sources that can differentiate into squamousepithelia, including both primary and immortalized keratinocytes.Sources of cells include keratinocytes and dermal fibroblasts biopsiedfrom humans and cavaderic donors (Auger et al, In Vitro Cell. Dev.Biol.—Animal 36:96-103; U.S. Pat. Nos. 5,968,546 and 5,693,332, each ofwhich is incorporated herein by reference), neonatal foreskins (Asbillet al., Pharm. Research 17(9): 1092-97 (2000); Meana et al., Burns24:621-30 (1998); U.S. Pat. Nos. 4,485,096; 6,039,760; and 5,536,656,each of which is incorporated herein by reference), and immortalizedkeratinocytes cell lines such as NM1 cells (Baden, In Vitro Cell. Dev.Biol. 23(3):205-213 (1987)), HaCaT cells (Boucamp et al., J. cell. Boil.106:761-771 (1988)); and NIKS® cells (Cell line BC-1-Ep/SL; U.S. Pat.No. 5,989,837, incorporated herein by reference; ATCC CRL-12191). Eachof the mentioned cell lines can be cultured or genetically modified inorder to produce a cell line capable of expressing or co-expressing thedesired protein(s). In particularly preferred embodiments, NIKS® cellsare utilized. The discovery of the novel NIKS® human keratinocyte cellline provides an opportunity to genetically engineer human keratinocyteswith non-viral vectors. A unique advantage of the NIKS® cells is thatthey are a consistent source of genetically-uniform, pathogen-free humankeratinocytes. For this reason, they are useful for the application ofgenetic engineering and genomic gene expression approaches to providehuman skin equivalents with enhanced properties over currently availableskin equivalents. NIKS® cells, identified and characterized at theUniversity of Wisconsin, are nontumorigenic, karyotypically stable, andexhibit normal growth and differentiation both in monolayer andorganotypic culture. NIKS® cells form fully stratified skin equivalentsin culture. These cultures are indistinguishable by all criteria testedthus far from organotypic cultures formed from primary humankeratinocytes. Unlike primary cells however, NIKS® cells exhibit anextended lifespan in monolayer culture. This provides an opportunity togenetically manipulate the cells and isolate new clones of cells withnew useful properties (Allen-Hoffmann et al., J. Invest. Dermatol.,114(3): 444-455 (2000)).

The NIKS® cells arose from the BC-1-Ep strain of human neonatal foreskinkeratinocytes isolated from an apparently normal male infant. In earlypassages, the BC-1-Ep cells exhibited no morphological or growthcharacteristics that were atypical for cultured normal humankeratinocytes. Cultivated BC-1-Ep cells exhibited stratification as wellas features of programmed cell death. To determine replicative lifespan,the BC-1-Ep cells were serially cultivated to senescence in standardkeratinocyte growth medium at a density of 3×105 cells per 100-mm dishand passaged at weekly intervals (approximately a 1:25 split). Bypassage 15, most keratinocytes in the population appeared senescent asjudged by the presence of numerous abortive colonies which exhibitedlarge, flat cells. However, at passage 16, keratinocytes exhibiting asmall cell size were evident. By passage 17, only the small-sizedkeratinocytes were present in the culture and no large, senescentkeratinocytes were evident. The resulting population of smallkeratinocytes that survived this putative crisis period appearedmorphologically uniform and produced colonies of keratinocytesexhibiting typical keratinocyte characteristics including cell-celladhesion and apparent squame production. The keratinocytes that survivedsenescence were serially cultivated at a density of 3×105 cells per100-mm dish. Typically the cultures reached a cell density ofapproximately 8×106 cells within 7 days. This stable rate of cell growthwas maintained through at least 59 passages, demonstrating that thecells had achieved immortality. The keratinocytes that emerged from theoriginal senescencing population are now termed NIKS®. The NIKS® cellline has been screened for the presence of proviral DNA sequences forHIV-1, HIV-2, EBV, CMV, HTLV-1, HTLV-2, HBV, HCV, B-19 parvovirus,HPV-16, SV40, HHV-6, HHV-7, HPV-18 and HPV-31 using either PCR orSouthern analysis. None of these viruses were detected.

Chromosomal analysis was performed on the parental BC-1-Ep cells atpassage 3 and NIKS® cells at passages 31 and 54. The parental BC-1-Epcells have a normal chromosomal complement of 46, XY. At passage 31, allNIKS cells contained 47 chromosomes with an extra isochromosome of thelong arm of chromosome 8. No other gross chromosomal abnormalities ormarker chromosomes were detected. The karyotype of the NIKS® cells hasbeen shown to be stable to at least passage 54.

The DNA fingerprints for the NIKS® cell line and the BC-1-Epkeratinocytes are identical at all twelve loci analyzed demonstratingthat the NIKS® cells arose from the parental BC-1-Ep population. Theodds of the NIKS® cell line having the parental BC-1-Ep DNA fingerprintby random chance is 4×10-16. The DNA fingerprints from three differentsources of human keratinocytes, ED-1-Ep, SCC4 and SCC13y are differentfrom the BC-1-Ep pattern. This data also shows that keratinocytesisolated from other humans, ED-1-Ep, SCC4, and SCC13y, are unrelated tothe BC-1-Ep cells or each other. The NIKS® DNA fingerprint data providesan unequivocal way to identify the NIKS® cell line.

Loss of p53 function is associated with an enhanced proliferativepotential and increased frequency of immortality in cultured cells. Thesequence of p53 in the NIKS® cells is identical to published p53sequences (GenBank accession number: M14695). In humans, p53 exists intwo predominant polymorphic forms distinguished by the amino acid atcodon 72. Both alleles of p53 in the NIKS® cells are wild-type and havethe sequence CGC at codon 72, which codes for an arginine. The othercommon form of p53 has a proline at this position. The entire sequenceof p53 in the NIKS® cells is identical to the BC-1-Ep progenitor cells.Rb was also found to be wild-type in NIKS® cells.

Anchorage-independent growth is highly correlated to tumorigenicity invivo. For this reason, the anchorage-independent growth characteristicsof NIKS® cells in agar or methylcellulose-containing medium wereinvestigated. NIKS® cells remained as single cells after 4 weeks ineither agar- or methylcellulose-containing medium. The assays werecontinued for a total of 8 weeks to detect slow growing variants of theNIKS® cells. None were observed.

To determine the tumorigenicity of the parental BC-1-Ep keratinocytesand the immortal NIKS® keratinocyte cell line, cells were injected intothe flanks of athymic nude mice. The human squamous cell carcinoma cellline, SCC4, was used as a positive control for tumor production in theseanimals. The injection of samples was designed such that animalsreceived SCC4 cells in one flank and either the parental BC-1-Epkeratinocytes or the NIKS® cells in the opposite flank. This injectionstrategy eliminated animal to animal variation in tumor production andconfirmed that the mice would support vigorous growth of tumorigeniccells. Neither the parental BC-1-Ep keratinocytes (passage 6) nor theNIKS® keratinocytes (passage 35) produced tumors in athymic nude mice.

NIKS® cells were analyzed for the ability to undergo differentiation inboth submerged culture and organotypic culture. Techniques fororganotypic culture are described in detail in the examples. Inparticularly preferred embodiments, the organotypically cultured skinequivalents of the present invention comprise a dermal equivalent formedfrom collagen or a similar material and fibroblasts. The keratinocytes,for example NIKS® cells or a combination of NIKS® cells and cells from apatient are seeded onto the dermal equivalent and form an epidermallayer characterized by squamous differentiation following theorganotypic culture process.

For cells in submerged culture, the formation of cornified envelopes wasmonitored as a marker of squamous differentiation. In cultured humankeratinocytes, early stages of cornified envelope assembly results inthe formation of an immature structure composed of involucrin,cystatin-a and other proteins, which represent the innermost third ofthe mature cornified envelope. Less than 2% of the keratinocytes fromthe adherent BC-1-Ep cells or the NIKS® cell line produce cornifiedenvelopes. This finding is consistent with previous studiesdemonstrating that actively growing, subconfluent keratinocytes produceless than 5% cornified envelopes. To determine whether the NIKS® cellline is capable of producing cornified envelopes when induced todifferentiate, the cells were removed from adherent culture andsuspended for 24 hours in medium made semi-solid with methylcellulose.Many aspects of terminal differentiation, including differentialexpression of keratins and cornified envelope formation can be triggeredin vitro by loss of keratinocyte cell-cell and cell-substratum adhesion.The NIKS® keratinocytes produced as many as and usually more cornifiedenvelopes than the parental keratinocytes. These findings demonstratethat the NIKS® keratinocytes are not defective in their ability toinitiate the formation of this cell type-specific differentiationstructure.

To confirm that the NIKS® keratinocytes can undergo squamousdifferentiation, the cells were cultivated in organotypic culture.Keratinocyte cultures grown on plastic substrata and submerged in mediumreplicate but exhibit limited differentiation. Specifically, humankeratinocytes become confluent and undergo limited stratificationproducing a sheet consisting of 3 or more layers of keratinocytes. Bylight and electron microscopy there are striking differences between thearchitecture of the multilayered sheets formed in submerged culture andintact human skin. In contrast, organotypic culturing techniques allowfor keratinocyte growth and differentiation under in vivo-likeconditions. Specifically, the cells adhere to a physiological substratumconsisting of dermal fibroblasts embedded within a fibrillar collagenbase. The organotypic culture is maintained at the air-medium interface.In this way, cells in the upper sheets are air-exposed while theproliferating basal cells remain closest to the gradient of nutrientsprovided by diffusion through the collagen gel. Under these conditions,correct tissue architecture is formed. Several characteristics of anormal differentiating epidermis are evident. In both the parental cellsand the NIKS® cell line a single layer of cuboidal basal cells rests atthe junction of the epidermis and the dermal equivalent. The roundedmorphology and high nuclear to cytoplasmic ratio is indicative of anactively dividing population of keratinocytes. In normal humanepidermis, as the basal cells divide they give rise to daughter cellsthat migrate upwards into the differentiating layers of the tissue. Thedaughter cells increase in size and become flattened and squamous.Eventually these cells enucleate and form cornified, keratinizedstructures. This normal differentiation process is evident in the upperlayers of both the parental cells and the NIKS® cells. The appearance offlattened squamous cells is evident in the upper epidermal layers anddemonstrates that stratification has occurred in the organotypiccultures. In the uppermost part of the organotypic cultures theenucleated squames peel off the top of the culture. To date, nohistological differences in differentiation at the light microscopelevel between the parental keratinocytes and the NIKS® keratinocyte cellline grown in organotypic culture have been observed.

To observe more detailed characteristics of the parental (passage 5) andNIKS® (passage 38) organotypic cultures and to confirm the histologicalobservations, samples were analyzed using electron microscopy. Parentalcells and the immortalized NIKS® human keratinocyte cell line wereharvested after 15 days in organotypic culture and sectionedperpendicular to the basal layer to show the extent of stratification.Both the parental cells and the NIKS® cell line undergo extensivestratification in organotypic culture and form structures that arecharacteristic of normal human epidermis. Abundant desmosomes are formedin organotypic cultures of parental cells and the NIKS® cell line. Theformation of a basal lamina and associated hemidesmosomes in the basalkeratinocyte layers of both the parental cells and the cell line wasalso noted.

Hemidesmosomes are specialized structures that increase adhesion of thekeratinocytes to the basal lamina and help maintain the integrity andstrength of the tissue. The presence of these structures was especiallyevident in areas where the parental cells or the NIKS® cells hadattached directly to the porous support. These findings are consistentwith earlier ultrastructural findings using human foreskin keratinocytescultured on a fibroblast- containing porous support. Analysis at boththe light and electron microscopic levels demonstrate that the NIKS®cell line in organotypic culture can stratify, differentiate, and formstructures such as desmosomes, basal lamina, and hemidesmosomes found innormal human epidermis.

In some embodiments, the tissues that are supported on the porousmembrane and enclosed with the container assembly are cryopreserved.Where this tissue is a skin equivalent, the cryopreserved skinequivalents are preferably storable at approximately −50 C, −60 C, −70C, −80 C or colder for an extended period of time such as greater than1, 2, 3, 4, 5 or 6 months and up to 12 or 24 months without asubstantial loss of viability.

In preferred embodiments, all steps of the cryopreservation processprior to product packaging are performed aseptically inside a Class 100biosafety cabinet in a Class 10,000 cleanroom. In some embodiments, thecryopreservation process comprises treating an organotypically culturedskin equivalent in a cryoprotectant solution. The organotypicallycultured skin equivalent is supported on a porous membrane of a tray ofthe present disclosure, and the tray is placed in a suitable vessel,such as a culture vessel or a tissue container assembly of the presentdisclosure. A suitable volume of cryoprotectant solution is added to thevessel to be in contact with the porous membrane, but not submerge thetissue, allowing cryoprotectant transfer into the tissue through itsbase. Certain embodiments of the present invention are not limited tothe use of any particular cryoprotectant. In some preferred embodiments,the cryoprotectant is glycerol. The cryoprotectant may be provided indifferent concentrations in the cryoprotectant solution. In someembodiments, the cryoprotectant is provided in a solution comprisingabout 20% or 21% to about 70% of the solution by volume, and morepreferably about 20% or 21% to about 45% of the solution by volume or37.5% to 62.5% of the solution by volume, or most preferably from about25% to 40% of the solution by volume or 42.5% to 57.5% of the solutionby volume, depending on the temperature. In some embodiments, thecryoprotectant solution preferably comprises about 32.5% v/v or about50% v/v cryoprotectant (e.g., glycerol). In some embodiments, thecryoprotectant is provided in a base medium solution. Suitable basemedium solutions include, but are not limited to, DMEM, Ham's F-10,Ham's F-12, DMEM/F-12, Medium 199, MEM and RPMI. In some embodiments,the base medium forms the remainder of the solution volume. In someembodiments, the cryoprotectant solution is buffered. Suitable buffersinclude, but are not limited to, HEPES, Tris, MOPS, and Trizma buffers.Buffering agents may be included at an amount to provide a bufferedsystem in the range of pH 7.0 to 7.4. In some preferred embodiments, thecryoprotectant solution is buffered with from about 5 mM to 15 mM HEPES,most preferably about 10 mM HEPES to a pH of about 7.0 to 7.4.

In some particularly preferred embodiments, treatment with thecryoprotectant solution is conducted in a single step. By “single step”it is meant that the cryoprotectant solution is not exchanged during theequilibration procedure as is common in the art. For example, thetreatment step is performed using a cryoprotectant solution with adefined concentration of cryoprotectant as opposed to a stepwiseequilibration procedure where several media changes with increasingconcentrations of cryoprotectant at each step. In some embodiments, thetreatment step is conducted at a reduced temperature. In preferredembodiments, the treatment step is conducted at from about 2 C to 8 C,while in other embodiments, the treatment step is conducted at roomtemperature, for example from about 15 C to 30 C. In some embodiments,the skin equivalent is incubated in the cryoprotectant solution forabout 10 to 60 minutes, preferably from about 20 to 30 minutes.

In some embodiments, the skin equivalent supported on the porousmembrane of the tray is frozen following treatment with thecryoprotectant solution, preferably after excess cryoprotectant solutionis removed from the skin equivalent, for example by aspirating thesolution or moving the treated skin equivalent to a fresh vessel (e.g.,a sterile culture vessel or a sterile tissue container assembly of thepresent disclosure). Accordingly, in some embodiments, the treated skinequivalent supported on the porous membrane of the tray is frozen byexposure to temperatures ranging from about −50 C to −100 C, and mostpreferably at about −80 C. In some preferred embodiments the tray withthe treated skin equivalent is simply placed in a bag or other vessel(e.g., a sterile culture vessel or a sterile tissue container assemblyof the present disclosure) and placed in a freezing unit such as a lowtemperature (e.g., −80° C. freezer) freezing unit. In contrast, it iscommon in the art to control the rate of freezing either by controllingthe temperature in the freezing unit or by placing the tissue to befrozen in a container that allows control of the rate of decrease intemperature.

In some embodiments, the cryopreserved skin equivalent is packaged forlong term storage. In some preferred embodiments, the skin equivalent,in its tray, is enclosed with the bottom and top containers as describedin detail above. Is some embodiments, the assembly containing the humanskin equivalent is sealed, preferably heat sealed in a sterile bag(e.g., a plastic or polymer bag) to provide a primary package. Theprimary package is then sealed inside a secondary bag, for example asecondary plastic, foil, or Mylar bag. The cryopreserved tissues of thepresent invention may preferably be stored at low temperature, fromabout −50 C to about −100 C or lower, preferably about −80 C. The skinequivalents may be preferably stored from about 1, 2, 3, 4, 5 or 6months and up to 12 or 24 months without a substantial loss ofviability.

In a preferred embodiment, an organotypically cultured skin equivalentin its tray, which is inserted into a sterile bottom container of thepresent disclosure, is treated with a cryoprotectant solution asdescribed above. Excess cryoprotectant solution is removed from the skinequivalent prior to freezing by aspirating the cryoprotectant solutionfrom the bottom container. The treated skin equivalent in its tray isthen enclosed with a sterile top container of the present disclosure,thereby forming a tissue container system. Alternatively, excesscryoprotectant solution is removed from the skin equivalent prior tofreezing by moving the tray with the treated skin equivalent to asecond, sterile bottom container of the present disclosure and thenenclosing the tray with a sterile top container of the presentdisclosure, thereby forming a tissue container system. The tissuecontainer system containing the treated human skin equivalent is thensealed, preferably heat sealed in a sterile bag (e.g., a plastic orpolymer bag) to provide a primary package. The primary package may besealed inside a secondary bag, for example a secondary plastic, foil, orMylar bag. The primary or secondary bag is then stored at lowtemperature, from about −50 C to about −100 C, preferably about −80 C.The skin equivalents may be stored from about 1, 2, 3, 4, 5 or 6 monthsand up to 12 or 24 months without a substantial loss of viability.

In another preferred embodiment, an organotypically cultured skinequivalent in its tray, which is placed in a culture vessel, is treatedwith a cryoprotectant solution as described above. Excess cryoprotectantsolution is removed from the skin equivalent prior to freezing by movingthe tray with the treated skin equivalent to a sterile bottom containerof the present disclosure and then enclosing the tray with a sterile topcontainer of the present disclosure, thereby forming a tissue containersystem. The tissue container system containing the treated human skinequivalent is then sealed, preferably heat sealed in a sterile bag(e.g., a plastic or polymer bag) to provide a primary package. Theprimary package may be sealed inside a secondary bag, for example asecondary plastic, foil, or Mylar bag, to produce a secondary package.The primary or secondary package is then stored at low temperature, fromabout −50 C to about −100 C, preferably about −80 C. The skinequivalents may be stored from about 1, 2, 3, 4, 5 or 6 months and up to12 or 24 months without a substantial loss of viability.

In some embodiments, the present invention provides a method of thawinga cryopreserved skin equivalent prior to application to a subject,comprising providing a cryopreserved tissue in the tissue containersystem as described above, removing the top tissue container to exposethe cryopreserved tissue, and filling the reservoir in the bottom tissuecontainer with a liquid medium under conditions that the cryopreservedtissue thaws to provide a thawed tissue, where the cryoprotectantcontained within the tissue is diluted into the liquid medium, leaving atissue that is substantially free of cryoprotectant. In otherembodiments, the method comprises removing a primary or secondarypackage containing a tissue container system comprising a cryopreservedtissue from a freezer or shipping container, removing the tissuecontainer system from the package(s), removing the top tissue containerto expose the cryopreserved tissue, and transferring the tray with thecryopreserved skin equivalent from the first tissue container into asecond tissue container that is sterile and staged in the sterile fieldand contains a liquid medium in the container reservoir, such that thetransferred cryopreserved tissue thaws to provide a thawed tissue andthe cryoprotectant contained within the tissue is diluted into theliquid medium. In some of the above embodiments, the liquid medium is atissue compatible solution, preferably a buffered solution. Suitabletissue compatible solutions include, but are not limited to, DMEM, Ham'sF-10, Ham's F-12, DMEM/F-12, Medium 199, MEM and RPMI. Suitable buffersinclude, but are not limited to, HEPES, Tris, MOPS, and Trizma buffers.Buffering agents may be included at an amount to provide a bufferedsystem in the range of pH 7.0 to 7.4. In still other embodiments, thetray with the cryopreserved skin equivalent is removed from the tissuecontainer and placed on an absorbent medium to remove thawedcryoprotectant solution from the skin equivalent. The absorbent mediummay be in any suitable, preferably sterile, vessel (e.g., a culturevessel or a fresh tissue container assembly). The present invention isnot limited to the use of a particular absorbent medium. Suitableabsorbent media include, but are not limited to, Telfa® pads, cellulosicpads (e.g., Whatman 1003-090 filter pads and Pall 70010 filter pads),gauze pads, and foam pads (e.g., Covidien 55544 hydrophilic foam pad).In some preferred embodiments, the absorbent medium is a Telfa® pad. Insome embodiments, the absorbent medium further comprises atissue-compatible solution. In some embodiments, the tissue compatiblesolution is a buffered solution. Suitable tissue compatible solutionsinclude, but are not limited to, DMEM, Ham's F-10, Ham's F-12,DMEM/F-12, Medium 199, MEM and RPMI. Suitable buffers include, but arenot limited to, HEPES, Tris, MOPS, and Trizma buffers. Buffering agentsmay be included at an amount to provide a buffered system in the rangeof pH 7.0 to 7.4.

It is contemplated that the cryopreserved skin equivalents of thepresent invention may be used therapeutically after thawing. In someembodiments, the cryopreserved skin substitute is used after thawing inwound closure and burn treatment applications. The use of autografts andallografts for the treatment of burns and wound closure is described inMyers et al., A. J. Surg. 170(1):75-83 (1995) and U.S. Pat. Nos.5,693,332; 5,658,331; and 6,039,760, each of which is incorporatedherein by reference. In some embodiments, the skin equivalents may beused in conjunction with dermal replacements such as DERMAGRAFT orINTEGRA. Accordingly, the present invention provides methods for woundclosure, including ulcers or wounds caused by burns, comprisingproviding a cryopreserved skin equivalent in a tissue container systemof the present disclosure, thawing the skin equivalent, and treating apatient suffering from a wound with the thawed skin equivalent underconditions such that the wound is closed.

In some embodiments, the skin equivalents are utilized to treat chronicskin wounds. Chronic skin wounds (e.g., venous ulcers, diabetic ulcers,pressure ulcers) are a serious problem. The healing of such a woundoften takes well over a year of treatment. Treatment options currentlyinclude dressings and debridement (use of chemicals or surgery to clearaway necrotic tissue), and/or antibiotics in the case of infection.These treatment options take extended periods of time and high levels ofpatient compliance. As such, a therapy that can increase apractitioner's success in healing chronic wounds and accelerate the rateof wound healing would meet an unmet need in the field. Accordingly, thepresent invention contemplates treatment of skin wounds withcryopreserved skin equivalents. In some embodiments, skin equivalentsare topically applied to wounds after thawing. In other embodiments,cryopreserved skin equivalents are used for application to partialthickness wounds after thawing. In other embodiments, cryopreserved skinequivalents are used to treat full thickness wounds after thawing. Inother embodiments, cryopreserved skin equivalents are used to treatnumerous types of internal wounds after thawing, including, but notlimited to, internal wounds of the mucous membranes that line thegastrointestinal tract, ulcerative colitis, and inflammation of mucousmembranes that may be caused by cancer therapies. In still otherembodiments, skin equivalents expressing host defense peptides orpro-angiogenic factors are used as a temporary or permanent wounddressing after thawing.

In still further embodiments, the cells are engineered to provideadditional therapeutic agents to a subject. The present invention is notlimited to the delivery of any particular therapeutic agent. Indeed, itis contemplated that a variety of therapeutic agents may be delivered tothe subject, including, but not limited to, enzymes, peptides, peptidehormones, other proteins, ribosomal RNA, ribozymes, small interferingRNA (siRNA) micro RNA (miRNA), and antisense RNA. In preferredembodiments, the agents are host defense peptides such as humanbeta-defensin 1, 2, or 3 or cathelicidin or other proteins such as VEGFand HIF-1 a, see, e.g., U.S. Pat. Nos. 7,674,291; 7,807,148; 7,915,042;7,988,959; and 8,092,531; each of which is incorporated herein byreference in its entirety. These therapeutic agents may be delivered fora variety of purposes, including but not limited to the purpose ofcorrecting genetic defects. In some particular preferred embodiments,the therapeutic agent is delivered for the purpose of detoxifying apatient with an inherited inborn error of metabolism (e.g.,aminoacidopathesis) in which the skin equivalent serves as wild-typetissue. It is contemplated that delivery of the therapeutic agentcorrects the defect. In some embodiments, the cells are transfected witha DNA construct encoding a therapeutic agent (e.g., insulin, clottingfactor IX, erythropoietin, etc.) and skin equivalents prepared fromtransfected cells are administered to the subject. The therapeutic agentis then delivered to the patient's bloodstream or other tissues from thegraft. In preferred embodiments, the nucleic acid encoding thetherapeutic agent is operably linked to a suitable promoter. The presentinvention is not limited to the use of any particular promoter. Indeed,the use of a variety of promoters is contemplated, including, but notlimited to, inducible, constitutive, tissue-specific, andkeratinocyte-specific promoters. In some embodiments, the nucleic acidencoding the therapeutic agent is introduced directly into thekeratinocytes (i.e., by electroporation, calcium phosphateco-precipitation, or liposome transfection). In other preferredembodiments, the nucleic acid encoding the therapeutic agent is providedas a vector and the vector is introduced into the keratinocytes bymethods known in the art. In some embodiments, the vector is an episomalvector such as a replicating plasmid. In other embodiments, the vectorintegrates into the genome of the keratinocytes. Examples of integratingvectors include, but are not limited to, retroviral vectors,adeno-associated virus vectors, non-replicating plasmid vectors andtransposon vectors

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); mM (millimolar); pM(micromolar); N (Normal); mol (moles); mmol (millimoles); pmol(micromoles); nmol (nanomoles); g (grams); mg (milligrams); lig(micrograms); ng (nanograms); 1 or L (liters); ml or mL (milliliters);μ1 or μL (microliters); cm (centimeters); mm (millimeters); pm(micrometers); nm (nanometers); C (degrees Centigrade); U (units), mU(milliunits); min. (minutes); sec. (seconds); % (percent); kb(kilobase); bp (base pair); PCR (polymerise chain reaction); BSA (bovineserum albumin); CFU (colony forming units); kGy (kiloGray); PVDF(polyvinylidine fluoride); BCA (bicinchoninic acid); SDS-PAGE (sodiumdodecyl sulfate polyacrylamide gel electrophoresis).

Example 1

StrataGraft® skin tissue is a living, full-thickness, allogeneic humanskin substitute that reproduces many of the structural and biologicalproperties of normal human skin. StrataGraft® skin tissue contains botha viable, fully-stratified epidermal layer derived from NIKS® cells,which are a consistent and well-characterized source of pathogen-freehuman keratinocyte progenitors, and a dermal layer containing normalhuman dermal fibroblasts (NHDF) embedded in a collagen-rich matrix.StrataGraft® skin tissue possesses excellent tensile strength andhandling characteristics that enable it to be meshed, stapled, andsutured similarly to human skin grafts. StrataGraft® also exhibitsbarrier function comparable to that of intact human skin and is capableof delivering bioactive molecules for wound bed conditioning and tissueregeneration. The physical and biological characteristics ofStrataGraft® skin tissue make it ideal for the treatment of a variety ofskin wounds.

The manufacturing process for StrataGraft® skin tissue encompasses threesequential cell and tissue culture processes. In Stage I of themanufacturing process, NIKS® keratinocytes are expanded in monolayercell culture. Concurrent with the NIKS® keratinocyte culture in Stage I,NHDF are expanded in monolayer culture and combined with purified type Icollagen and culture medium and allowed to gel to form the cellularizeddermal equivalent (DE). Alternatively, NHDF are seeded into Transwell®inserts (Corning) and allowed to proliferate and secrete and assembleextracellular matrix molecules into a simplified dermal equivalent. InStage II, NIKS® keratinocytes are seeded onto the surface of the DE andcultured under submerged conditions for two days to promote completeepithelialization of the DE surface. The tissue is then lifted to theair-liquid interface in Stage III, where it is maintained for 18 days ina controlled, low humidity environment to promote tissue maturation. Theskin equivalents are generally prepared as described in U.S. Pat. Nos.7,674,291; 7,807,148; 7,915,042; 7,988,959; 8,092,531; and U.S. Pat.Publ. 20140271583; each of which is incorporated herein by reference inits entirety.

Example 2

This example describes improved cryopreservation methods for human skinequivalents utilizing a pre-freeze treatment step with cryopreservationsolutions containing 32.5% or 50% glycerol at room temperature and isdescribed in co-pending U.S. Pat. Publ. 20140271583, which isincorporated by reference herein in its entirety. The general productionprocess is unchanged from the current method described previously. Atthe end of the production process, the tissues are treated andcryopreserved as follows.

Parameter Operating Range Cryoprotectant formulation 32.5% (v/v)glycerol DMEM (1X) 10 mM HEPES (pH 7.0 to 7.4); or 50% (v/v) glycerolDMEM (1X) 10 mM HEPES (pH 7.0 to 7.4) Pre-freeze cryoprotectant Roomtemperature incubation temperature Pre-freeze cryoprotectant 15-45minutes incubation time Freeze method Direct transfer to −80 C. freezerStorage temperature −70 to −90 C. Shipping conditions Overnight deliveryon dry ice

All steps of the cryopreservation process prior to the final productpackaging step are performed aseptically inside a Class 100 biosafetycabinet in a Class 10,000 cleanroom. The specific volumes and dishesdescribed in this example are applicable to tissues generated in theprevious circular, 44 cm² format, not the larger rectangular format ofthe current disclosure.

Step 1—Dispense 20 ml of cryoprotectant solution to 100 mm culturedishes.

Step 2—Transfer Transwell® inserts containing StrataGraft® tissues intoindividual dishes containing cryoprotectant solution. Incubate tissues15-45 minutes in cryoprotectant solution.

Step 3—Transfer Transwell® inserts containing treated StrataGraft®tissues to new sterile 100 mm culture dishes containing final productlabel so that the tissue rests on the bottom of the culture dish. Excesscryoprotectant is allowed to drain from the skin equivalent to provide atreated skin equivalent that is substantially free of excesscryoprotectant on the exterior surfaces of the skin equivalent.

Step 4—Heat-seal 100 mm culture dishes in clear, sterile bags. Placeprimary package into secondary Mylar bag and heat-seal.

Step 5—Remove the packaged StrataGraft® tissues from cleanroom andtransfer tissues to an ultralow freezer (−70° C. to −90° C.). Placetissues in a pre-cooled rack in the freezer that allows unrestrictedairflow to the top and bottom of the packaged tissues to ensure uniformand rapid cooling. Leave tissues undisturbed overnight during thefreezing process.

Cryopreserved tissues were thawed at room temperature for 10 minutes,transferred to a hold chamber containing Telfa® pads saturated with 40ml of HEPES-buffered culture medium that had been warmed to roomtemperature (RT), and held at RT for 15 to 20 minutes. Tissues weretransferred to a culture dish containing 90 ml of SMO1 medium andreturned to culture overnight. Tissues were analyzed for viability afterovernight re-culture. Tissues treated with 32.5% glycerol at roomtemperature for 15 to 45 minutes had acceptable post-thaw viability.Tissues treated with 50% glycerol at room temperature for 15 minutesalso had acceptable viability; however, tissues treated with 50%glycerol at room temperature for 45 minutes had unacceptable viability.

Example 3

This study was performed to evaluate the performance of productpackaging plasticware, which is a tissue container assembly of thepresent disclosure, for use as packaging for cryopreserved StrataGraft®tissues. The study evaluated three independent lots of rectangular, 100cm² StrataGraft® tissues comparing tissues packaged in the Transwell®growth chamber and those packaged in the tissue containers describedherein. For each batch, post-thaw properties of tissues packaged in thetissue containers of the instant invention were evaluated followingdifferent hold conditions and compared to those of control tissues usingcurrent packaging and thaw/hold procedures. The results of this studydemonstrated that tissue containers of the instant invention aresuitable for use in transporting and thawing cryopreserved StrataGraft®tissues and that acceptable thawing can be achieved in the sterile fieldwithout use of a Telfa® pad.

StrataGraft® skin tissues are produced in batches of 100 cm²StrataGraft® skin tissues. This larger tissue format and increase inbatch sizes put an added emphasis on efficient storage and shipment ofthe skin tissues. To address that issue, plasticware tissue containerswere designed which reduce the volume of the final packaged product by60% compared to packaging in the Transwell® growth chamber as disclosedin copending U.S. Pat. Publ. 20140271583. In this example, thispackaging is introduced into the process following cryoprotectanttreatment, immediately before the product is sealed in the foil pouchand transferred to an ultracold freezer for long-term storage. Thetissue containers of the instant invention were designed with a 0.75 mmdeep reservoir below tissue that can be flooded with hold solution. Thisdesign allows the packaging to be used as a post-thaw hold container,which simplifies the preparation of StrataGraft® tissue for clinical useby eliminating the need for a separate hold basin.

This experiment evaluated the post-thaw properties of StrataGraft® skintissues from three batches, and frozen in either a Transwell® growthchamber or in the tissue containers of the instant invention. Inaddition, this study evaluated post-thaw hold procedures performed inthe tissues containers of the instant invention without the use ofTelfa® pads, compared to control hold conditions performed in basinscontaining Telfa® pads.

Pre-Freeze Thaw Hold Hold Hold Group Treatment Packaging ConditionChamber Solution Condition 1 37.5% Transwell ® 10 min DeRoyal 250 mLHold 15-20 min glycerol Growth at RT Basin Solution at RT 20 min at RTChamber (2-Telfa) Warmed to 35-39 ° C. 2 Tissue 3 container Tissue 15 mLassembly container Hold Solution assembly (no Warmed to Telfa ®) 35-39 °C.

Batches of 20 rectangular, 100 cm² StrataGraft® skin tissues wereproduced using Stratatech's standard processes. Briefly, NIKS® cells andnormal human dermal fibroblasts (NHDF) were expanded in monolayerculture. NHDF were thawed and expanded in monolayer. Followingexpansion, the NHDF cells were harvested and mixed into a type Icollagen solution, dispensed to 100 cm² rectangular trays of the presentdisclosure (tissue-culture treated polycarbonate membrane, nominalthickness of about 10 microns, nominal pore size of about 0.4 microns),and gelled to create the dermal equivalent layer (DE). After gelling,the DE was submerged in media in a growth chamber and cultured for fivedays prior to the NIKS® seed. NIKS® were thawed, expanded, and thenharvested and seeded onto DE surfaces. Tissues were maintained insubmerged culture for two days to allow for attachment and proliferationof NIKS® over the DE surface and then cultured at the air-liquidinterface for 18 days to enable complete epidermal differentiation.Transfers of media, NHDF/collagen mixture, and NIKS® suspension to thetrays and Transwell® growth chambers were performed using peristalticpumps.

At the end of the production process, culture media was aspirated andtissues were treated in the Transwell® growth chamber with 50 mL ofcryopreservation solution containing 37.5% glycerol for 20 minutes atroom temperature (RT) whilst still supported on the membrane of thetray. At the end of treatment, the trays containing the nine tissuesdesignated for this experiment were removed from the excesscryopreservation solution and packaged into one of two packagingconfigurations: 1) three tissues were kept in the Transwell® growthchamber in the high position and sealed inside of 7.875″×12″ foilpouches (Group 1); and 2) six tissues were transferred to sterile tissuecontainers of the instant invention and sealed inside 6.75″×10.25″ foilpeel pouches (Group 2 and Group 3, n=3 per group). At the end ofpackaging, all packaged tissues were transferred to an ultracold freezerand stored at −70 to −90° C. until analysis.

Group 1 and Group 2 tissues were then thawed using previouslyestablished procedures that utilized an absorbent medium (e.g., Telfa®pad). Group 3 tissues were thawed using a simplified hold procedure.Briefly, Group 3 cryopreserved tissues were thawed at room temperaturefor 10 minutes in the tissue container in which the tissue was frozen,the bottom tissue containers were then flooded with hold solution (15 mlof HEPES-buffered culture medium that had been warmed to 35-39 C) andheld at room temperature for 15 to 20 minutes. Following the post-thawhold, tissues from all groups were transferred to new rectangular growthchambers containing SM01 and re-cultured for 22 to 26 hours

Tissues were evaluated for appearance, barrier function, viability,histology, and VEGF secretion in the conditioned media. In addition,whole tissue MTT staining was performed to evaluate uniformity of thetissue viability. The results of tissues frozen in the tissue containersof the instant invention (groups 2 and 3) were compared to those of thecontrol group.

The results of this study demonstrate that use of the tissue containersof the instant invention does not affect the properties of cryopreservedStrataGraft® tissues. Tissues packaged in the two configurations andthawed/held using the previously established procedures (Groups 1 and 2)had comparable appearance, histology, viability, and barrier function,and VEGF secretion. The tissue containers of the instant invention alsoshowed promising results for use in a simplified hold procedure. Tissuespackaged and kept in tissue containers of the instant invention for thepost-thaw hold (Group 3) had similar properties to both other groups.Tissue appearance, histology, VEGF secretion, and barrier function werenot significantly different than control tissues (Group 1); viabilityshowed a modest (−10%), but statistically significant (p<0.05),reduction compared to controls, while still easily exceeding theestablished lot release criterion. MTT staining patterns of tissues fromall groups were comparable, with qualitatively consistent stainingacross the tissue surfaces. See FIGS. 5, 6 and 7.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in tissueculture, molecular biology, biochemistry, or related fields are intendedto be within the scope of the following claims.

1. A kit comprising: a sterile tissue container system comprising:substantially identical top and bottom sterile tissue containers; a traycomprising a porous bottom surface; and an organotypic skin substitutesupported on the porous bottom surface of the tray; a sterile packagecontaining the sterile tissue container system; and a tissue compatiblesolution.
 2. The kit of claim 1, wherein each of the top and bottomsterile tissue containers comprise: a perimeter wall comprising a flangeextending therefrom; a substantially planar bottom surface, theperimeter wall surrounding the bottom surface defining a dish having adish length and a dish width; at least one ridge projecting from theperimeter wall above the flange, the at least one ridge having a ridgelength and a ridge width; and at least one space recessed below theflange on the perimeter wall, the recessed space having a recess lengthand a recess width.
 3. The kit of claim 2, wherein the bottom surfacehas a perimeter defined by the perimeter wall and comprising a perimeterledge extending around the perimeter to provide a reservoir defined bythe perimeter ledge and the bottom surface.
 4. The kit of claim 2,wherein the perimeter wall has a male end and a female end opposite themale end along the dish length, wherein the at least one ridge islocated at the male end of the perimeter wall and the at least one spaceis located at the female end of the perimeter wall.
 5. The kit of claim4, wherein the flange comprises one or more tabs extending from the maleend of the perimeter wall.
 6. The kit of claim 5, wherein the flangecomprises one or more tabs extending from the female end of theperimeter wall.
 7. The kit of claim 4, wherein the ridge has a proximalend and the proximal end of the ridge has one or more indents therein.8. The kit of claim 4, wherein when the top and bottom sterile tissuecontainers are assembled the flanges of the top and bottom containerscontact one another.
 9. The kit of claim 8, wherein the flange comprisesone or more tabs extending from the male end of the perimeter wall andone or more tabs extending from the female end of the perimeter wall.10. The kit of claim 9, wherein when the top and bottom sterile tissuecontainers are assembled, the one or more tabs extending from the maleend of the perimeter wall of the bottom sterile tissue container do notoverlap with the one or more tabs extending from the female end of theperimeter wall of the top sterile tissue container.
 11. The kit of claim4, wherein when the top sterile tissue container is rotated and placedfacing the bottom sterile tissue container, the at least one space onthe female end of the bottom sterile tissue container releasablyreceives the at least one ridge on the male end of the top steriletissue container, thereby forming an enclosed container.
 12. The kit ofclaim 1, wherein the sterile tissue container is formed from a medicalgrade plastic.
 13. The kit of claim 1, wherein the porous bottom surfaceof the tray is a porous membrane.
 14. The kit of claim 13, wherein theporous membrane comprises a semi-permeable polymer film or asemi-permeable track-etched polymer film.
 15. The kit of claim 14,wherein the porous membrane comprises polyethylene terephthalate,polyester, polycarbonate, or combinations thereof.
 16. The kit of claim13, wherein the porous membrane is plasma treated to improve cellattachment.
 17. The kit of claim 13, wherein the porous membrane has athickness of about 5 microns to about 20 microns.
 18. The kit of claim13, wherein the porous membrane comprises pores with a pore size ofabout 0.1 micron to about 10 microns.
 19. The kit of claim 13, whereinthe porous membrane has a pore density of about 1×10⁵ and about 1×10⁸pores per square centimeter.
 20. The kit of claim 1, wherein the trayfurther comprises one or more tray tabs.
 21. The kit of claim 1, whereinthe organotypic skin substitute is cryopreserved.
 22. The kit of claim1, further comprising an absorbent medium.
 23. The kit of claim 22,wherein the absorbent medium is selected from the group consisting ofTelfa pads, cellulosic pads, gauze pads, and foam pads.
 24. The kit ofclaim 1, wherein the tissue compatible solution is a buffered solutionin the range of pH 7.0 to 7.4.
 25. The kit of claim 24, wherein thetissue compatible solution comprises DMEM, Ham's F-10, Ham's F-12,DMEM/F-12, Medium 199, MEM, RPMI, HEPES, Tris, MOPS, Trizma buffer, orcombinations thereof.
 26. The kit of claim 1, wherein the sterilepackage comprises a heat sealed sterile bag.
 27. The kit of claim 26,wherein the sterile bag is a plastic or polymer bag.
 28. The kit ofclaim 1, wherein the sterile package is sealed inside a secondary bag.29. The kit of claim 28, wherein the secondary bag comprises plastic,foil, or Mylar.